Human rotavirus in Iran; molecular epidemiology, genetic diversity and recent updates on vaccine advances.

Human rotavirus is the predominant pathogen causing gastroenteritis in infants and children younger than 5 years of age globally. Before introduction and implementation of rotavirus vaccine, more than frothy percent of all caused acute gastroenteritis hospitalization and nearly half a million deaths per year was occurred due to Rotavirus infection mostly in the low-income countries. Rotaviruses are divided in G and P genotypes, based on two genomic segments' nucleotide sequences VP7 and VP4, respectively. Currently, 27 G and 37 P types have been described; among them G1 to G4 and G9 and P[8], P[4], and P[6] genotypes are the most prevalent circulating rotavirus strains globally. Molecular epidemiological surveys revealed that G1P[8] is the predominant genotype in Iran, although other genotypes have also been reported. Rotavirus vaccine was recommended by the World Health Organization as a necessary part of national childhood immunization programs in 2009. Rotarix (monovalent) and RotaTeq (pantavalent) are two oral vaccines that have been available in more than one hundred countries around the world to control the viral infection and reduce the cases of diarrheal diseases. This article provides a review of frequency, molecular epidemiology and current situation of Rotavirus genetic diversity Iran. In addition, recent advances in rotavirus vaccine research are discussed.

rotavirus-associated mortality occurs generally in South Asia and Africa. Mortality is unusual in developed countries, however when it comes to society impact diarrhea has a noticeable effect in terms of healthcare and medical costs (4)(5)(6)(7)(8).
In developing regions, because of insufficient domestic sanitation and areas with inexistent access to the healthcare infrastructure, the average age of early exposure to RV is around 6 to 9 months old. In the developed world, where public health standards are considerably satisfactory, the average age of RV primary infection may occasionally be delayed until the age of 2-5 years. Acute gastroenteritis is an extremely common disease which often starts with fever, nausea and vomiting and followed by severe abdominal pain and cramps and frequent watery diarrhea. Checking the patient's history is important for diagnosis of AGE. When diarrhea occurs with vomiting, it may induce serious condition of dehydration. The best clinical indicators of dehydration include thirst, becoming irritable, weakness and fatigue, lethargy, dry mouth and tongue and also dry skins. Dehydration is an important complication of rotavirus, and this situation can lower blood volume, collapse and finally death (9)(10)(11).

Viral Structure
Rotaviruses are a genus of double-stranded RNA viruses of Reoviridae family. The viral architecture is consisted of a non-enveloped icosahedral capsid as a triple-layered particle (TLP). By electron microscopy analysis, TLPs look similar to a wheel, and this appearance is the main reason for the title "Rotavirus" (12,13). The capsid firmly wraps viral genome that is comprised of 11 segments of double-stranded RNA. The segments encode 6 structural viral products (VP1 to VP4, VP6 and VP7) and 5 or 6 non-structural proteins (NSP1 to NSP6) (9,(14)(15)(16)(17). Each double-stranded RNA segment encodes a single structural (VP1 to VP7) or nonstructural proteins (NSP1 to NSP4) other than segment 11, that encodes for NSP5 and NSP6 (1). The structural proteins can constitute rotavirus virion. The nonstructural proteins, are only involved in cells infected by rotavirus (14) . The inner and intermediate layers of the capsid are composed consequently of VP2 and VP6 structural protein, the outer part of the capsid is composed of VP4 and VP7 structural proteins which consist of neutralizing antigens that are classified into G and P serotypes, respectively via neutralization tests. In addition to these serotypes, genotypes G and P, based on the different genes of VP7 and VP4 have been defined to at least 27 G-types and 37 P-types. Recently, in most location around the word including Iran, G1P [8], G2P [4], G4P [8], G3P [8], and G9P [8] are common source of about 90% of all human infections associated with rotavirus , prevalence of G1P [8] is the highest among the other genotypes (8,9,15,18,19). During the first occurrence of rotavirus infection, rotaviruses are shed continuously for several days in high concentrations (>10¹² particles/gram) in patients' stool and vomit. Transmission occurs mainly by the fecal-oral route in direct manner from person to person, or in indirect manner through contaminated fomites (19).

Laboratory diagnosis
Diagnosis of etiological rotavirus gastroenteritis needs laboratory confirmation. Different diagnostic assays are commercially accessible: enzyme immunoassays for detecting antigens of rotavirus in stool specimens in direct manner are used generally, despite of the less sensitive, but rapid and easy-to-use test strips and latex agglutination method. Reverse transcription polymerase chain reaction (RT-PCR), that is very sensitive in diagnosis low concentrations of rotavirus in stool specimens, is also used for identification and further differentiation (19,20).

Epidemiology
Many studies performed to investigate rotavirus epidemiology in the world. Several researchers determined and reported the rates of rotavirus related infections in Latin America (30%), Europe (40%), and Africa and Middle East (34%-40%) (21)(22)(23)(24) In this recent study 95% of rotaviruses were determined as G types and 85% as P type. The frequency of P type was P(8) (74%), P(4) (11%), (15%) were P-non-typeable respectively, and the frequency of G type was G1 82%, G2 13%, and 5% were G-non-typeable (30 study to determine the role of rotavirus in children younger than 5 years suffering from acute gastroenteritis in two children medical center and one general hospital in Tehran. In 15.3% of children with acute diarrhea rotavirus antigen was diagnosed with highest incidence in children between 6-12 months of age. Rotavirus infection occurred with lower frequency in breast fed infants than bottle fed. The rate of detection was higher in the spring than summer, although the rate of hospitalization was the highest in winter (37). Samarbafzadeh et al. conducted a study in Ahwaz (city of Iran) from November 2001 to March 2002 in inpatient and outpatient children between 1 to 24 months old. Rotavirus was reported 26.3% of outpatients and 36.5% of hospitalized patients. In this study population, the frequency of rotavirus was 29.5% with highest incidence in children between 7 to 12 months old that showed a potential link between age and rate of rotavirus infection (38). Kazemi  In this study the percentage of the most common genotype was for G types 60.0% for nontypeable 12.50% for G2 12.5 % for G4 10.05 % and for G1 5% . The highest prevalence of rotavirus was in children less than 24 months of age (69%). The highest incidence of rotavirus infection was in summer (52.5%) and the lowest in winter (7.5%) and 72.91% of rotavirus infections were occurred in children less than 2 years of age (50). Khoshdel et al. conducted a study in children 6-60 months of age who were admitted in hospital due to diseases except diarrhea during December 2010 and October 2011 in Shahrekord, Iran. In this study the incidence of rotavirus related infection was 30% and the prevalence of genotypes was G1 (20%) , G9 (20%) , mixed genotypes: G1+G9 (13.3%), G1+G4 (6.7%), and G1+G8 (3.3%) , G1+G3 (3.3%), respectively (51). Rahbarimanesh

Rotavirus vaccine
Vaccines are an efficient and accessible proceeding for safeguarding from rotavirus complications and also for prevention of rotavirus diarrhea. According to WHO estimation, globally 23% of susceptible newborns had rotavirus vaccine in 2015 with coverage approximately fourfold lower than the estimated overall coverage for polio and diphtheria-tetanus-pertussis vaccines (86%) (20,61,62). From May 2016, a group of countries has entered rotavirus vaccine into their internal immunization policies. Presently, 3 kinds of vaccine including live, oral, attenuated (rotavirus strains of human and/or animal origin that can replicate in the human intestinal epithelial cells) are available for Rotavirus (62). Generally, there is two oral live attenuated rotavirus vaccines that are presented globally including the monovalent (RV1) and the pentavalent (RV5) vaccines (20). The RV1 (lyophilized and liquid) also known as RV1 is an oral vaccine that originated from a G1P (8) strain that was isolated from a case of infantile gastroenteritis. Another type of oral vaccines for Rotavirus is the RV5 which includes five reassortant rotaviruses developed from human and bovine derived rotavirus strains. Many challenges remain until we assess to the global effective rotavirus vaccines that can prevent severe and fatal cases of rotavirus infection in children. These second-generation rotavirus vaccines, RotaTeq (RV5) and RotarixTM (RV1), have been permitted in over 100 countries. Despite the high efficacy determined by the vaccines in studies in developed countries, the WHO Strategic Advisory Group of Experts (SAGE) on immunization, postponed making a recommendation for international practice in 2006, awaiting the accessibility of vaccine data survey from developing countries, including some African and Asian countries. Other additional pieces of evidence have found on live oral vaccines effectiveness variations between various populations, with efficacy being lower in developing country populations with the maximum rate of disease. Although both vaccines were confirmed to be effective in preventing severe rotavirus disease due to homotypic and heterotypic rotavirus vaccine strains, long-term monitoring is needed to assess possible selection pressure. Additionally, as both available vaccines are developed from only a limited number of strains of rotavirus, there have been considerations that they would not adequately protect from serious infection induced by non-vaccine rotavirus strains (20,62,63 [8] was the predominant (34.8%), followed by G1P [8] and G2P [4] (each of them 13%), the rates of other genotypes were 8.7% for G9P [6], 4.3% for each of G4P [8] and G1P [6], 21.7% for mixed genotypes G1G9P [8] (13%), G1G4P [6] (4.3%), G2G9P [8] (4.3%) (Figure 1). During 2007 to 2008 the prevalence of rotavirus was 19.3% and G2P [4] was the predominant (35.1%), followed by G1P [8] (27%) the rates of other genotypes were 24.3% for G9P [8], 2.7% for each of G2P [6], G2P [8] and G12P [6] were not identified and in terms of P types. 50% were not identified and 25% were P [8] and 21% were P(6) and 4% were P (4). In 2006 in terms of G types, 68% of rotavirus positive cases were G2, 4% were G9 and 28% were not identified and in terms of P types 56% were P (4), 8% were P (8), 8% were P (6) and 50% were not identified.

Conclusion
Rotavirus is one of the most important infectious agents that cause the mortality of infants and children less than 5 years old worldwide. After approval, wide application and introduction of rotavirus vaccine into national immunization programs in developed countries, the prevalence of RV infection and mortality rate have been decreased significantly. However, it is necessary to continue nationwide surveys to control and monitor any cases of RT gastroenteritis to elucidate the remaining.